Clostridium difficile, a Gram positive anaerobic spore-forming bacillus is an etiologic agent of antibiotic associated diarrhea (AAD) and colitis (AAC). The symptoms of the disease range from mild diarrhea to fulminant and life-threatening pseudomembranous colitis (PMC). Antibiotic therapy can disrupt the normal intestinal microflora. Destruction of the normal flora results in a condition in which C. difficile can spores of C. difficile can germinate and the organism can grow and produce disease causing toxins. C. difficile causes about 25% of antibiotic-associated diarrheas, however, it is almost always the causative agent of PMC (Lyerly, D. M. and T. D. Wilkins, in Infections of the Gastrointestinal Tract, Chapter 58, pages 867-891, (Raven Press, Ltd, New York 1995)). Additionally, C. difficile is frequently identified as a causative agent of nosocomial infectious diarrheas, particularly in older or immuno-compromised patients (U.S. Pat. No. 4,863,852 (Wilkins et al.) (1989)).
Disease caused by C. difficile is due to two enteric toxins A and B produced by toxigenic strains (U.S. Pat. No. 5,098,826 (Wilkins et al.) (1992)). Toxin A is an enterotoxin with minimal cytotoxic activity, whereas toxin B is a potent cytotoxin but has limited enterotoxic activity. The extensive damage to the intestinal mucosa is attributable to the action of toxin A, however, toxins A and B act synergistically in the intestine.
The genetic sequences encoding both toxigenic proteins A and B, the largest known bacterial toxins, with molecular weights of 308,000 and 269,000, respectively, have been elucidated (Moncrief et al., Infect. Immun. 65:1105-1108 (1997); Barroso et al., Nucl. Acids Res. 18:4004 (1990); Dove et al. Infect. Immun. 58:480-488 (1990)). Because of the degree of similarity when conserved substitutions are considered, these toxins are thought to have arisen from gene duplication. The proteins share a number of similar structural features with one another. For example, both proteins possess a putative nucleotide binding site, a central hydrophobic region, four conserved cysteines and a long series of repeating units at their carboxyl ends. The repeating units of toxin A, particularly, are immunodominant and are responsible for binding to type 2 core carbohydrate antigens on the surface of the intestinal epithelium (Krivan et al., Infect. Immun. 53:573-581 (1986); Tucker, K. and T. D. Wilkins, Infect. Immun. 59:73-78 (1991)).
The toxins share a similar molecular mechanism of action involving the covalent modification of Rho proteins. Rho proteins are small molecular weight effector proteins that have a number of cellular functions including maintaining the organization of the cytoskeleton. The covalent modification of Rho proteins is due to glucosyltransferase activity of the toxins. A glucose moiety is added to Rho using UDP-glucose as a co-substrate (Just et al. Nature 375:500-503 (1995), Just et al. J. Biol. Chem 270:13932-13939 (1995)). The glucosyltransferase activity has been localized to approximately the initial 25% of the amino acid sequence of each of these toxins (Hofmann et al. J. Biol. Chem. 272:11074-11078 (1997), Faust and Song, Biochem. Biophys. Res. Commun. 251:100-105 (1998)) leaving a large portion of the toxins, including the repeating units, that do not participate in the enzymatic activity responsible for cytotoxicity.
The toxin A protein comprises 31 contiguous repeating units (rARU ) and may contain multiple T cell epitopes (Dove et al. Infect. Immun. 58:480-488 (1990). The repeating units are defined as class I repeats and class II. rARU may be uniquely suited for use in inducing T cell-dependent response to an antigen. The sequence of each unit is similar but not identical. These features along with its usefulness in eliciting toxin A neutralizing antibodies make rARU a novel candidate as a carrier protein.
The toxin B repeating units have similar features to those of rARU. Like rARU, the recombinant toxin B repeating units (rBRU) are relatively large (˜70 kDa) and are composed of contiguous repeats of similar amino acid sequences (Barroso et al. Nucleic Acids Res. 18:4004 (1990); Eichel-Streiber et al. Gene 96:107-113 (1992)). Less is known about this portion of toxin B than the binding domain of toxin A.
Thomas et al (U.S. Pat. No. 5,919,463 (1999)) disclose C. difficile toxin A or toxin B or certain fragments thereof as mucosal adjuvants intranasally administered to stimulate an immune response to an antigen (e.g., Helicobacter pylori urease, ovalbumin (OVA), or keyhole limpet hemocyanin (KLH)). However, Thomas does not teach the use of such adjuvant for protection against strains of C. difficile. Lyerly et al. Current Microbiology 21:29-32 (1990) considered at a smaller recombinant fragment from the toxin A repeats in hamster protection assays. However, these data suggest at best only a very weak or partial protection from strains of C. difficile, whereas the present invention demonstrates the use of C. difficile toxin repeating units that provide a clear immunogenic response and at higher levels, which afford protection against C. difficile. 
Even were one to consider rARU and rBRU as candidate proteins for conjugate vaccines, the production of such proteins presents certain challenges. There are methods for the production of toxin A and antibodies elicited thereto (U.S. Pat. No. 4,530,833 (Wilkins et al.)(1985); U.S. Pat. No. 4,533,630 (Wilkins et al.)(1985); and U.S. Pat. No. 4,879,218 (Wilkins et al.)(1989)). There are significant difficulties in producing sufficient quantities of the C. difficile toxin A and toxin B proteins. These methods are generally cumbersome and expensive. However, the present invention provides for the construction and recombinant expression of a nontoxic truncated portions or fragments of C. difficile toxin A and toxin B in strains of E. coli. Such methods are more effective and commercially feasible for the production of sufficient quantities of a protein molecule for raising humoral immunogenicity to antigens.
Part of the difficulty that the present invention overcomes concerns the fact that large proteins are difficult to express at high levels in E. coli. Further, an unusually high content of AT in these clostridial gene sequences (i.e., AT-rich) makes them particularly difficult to express at high levels (Makoff et al. Bio/Technology 7:1043-1046 (1989)). It has been reported that expression difficulties are often encountered when large (i.e., greater than 100 kd) fragments are expressed in E. coli. A number of expression constructs containing smaller fragments of the toxin A gene have been constructed, to determine if small regions of the gene can be expressed to high levels without extensive protein degradation. In all cases, it was reported that higher levels of intact, full length fusion proteins were observed rather than the larger recombinant fragments (Kink et al., U.S. Pat. No. 5,736,139; see: Example 11(c)). It has been further reported that AT-rich genes contain rare codons that are thought to interfere with their high-level expression in E. coli (Makoff et al. Nucleic Acids Research 17:10191-10202). The present invention provides for methods to produce genes that are both large and AT-rich and immunogenic compositions thereof. For example, the toxin A repeating units are approximately 98 kDa and the gene sequence has an AT content of approximately 70% that is far above the approximately 50% AT content of the E. coli genome. The present invention provides for methods of expressing AT-rich genes (including very large ones) at high levels in E. coli without changing the rare codons or supplying rare tRNA.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. Further, all documents referred to throughout this application are incorporated in their entirety by reference herein.